Szilvia Déri1,2, János Borbás3, Teodóra Hartai1, Lidia Hategan3, Beáta Csányi3, Ádám Visnyovszki1, Tamara Madácsy4, József Maléth4, Zoltán Hegedűs5,6, István Nagy7,8, Rohit Arora9, Alain J Labro10,11, László Környei12, András Varró1,2,13, Róbert Sepp3, Balázs Ördög1. 1. Department of Pharmacology and Pharmacotherapy, University of Szeged, Dóm tér 12, PO Box 427, Szeged 6720, Hungary. 2. Department of Pharmacology and Pharmacotherapy, Interdisciplinary Excellence Centre, University of Szeged, Dóm tér 12, 6720 Szeged, Hungary. 3. 2nd Department of Internal Medicine and Cardiology Centre, University of Szeged, Semmelweis u. 8, 6725 Szeged, Hungary. 4. 1st Department of Internal Medicine, University of Szeged, Korányi fasor 8-10, 6720 Szeged, Hungary, Hungary. 5. Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, Temesvári krt. 62, 6726 Szeged, Hungary. 6. Department of Biochemistry and Medical Chemistry, University of Pécs, Szigeti út 12, 7624 Pécs, Hungary. 7. Institute of Biochemistry, Biological Research Centre the Hungarian Academy of Sciences, Temesvári krt. 62, 6726 Szeged, Hungary. 8. Seqomics Biotechnology Ltd, Vállalkozók útja 7, 6782 Mórahalom, Hungary. 9. Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium. 10. Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium. 11. Department of Basic Medical Sciences, University of Ghent, C. Heymanslaan 10, 9000 Ghent, Belgium. 12. Gottsegen György National Institute of Cardiology, Haller u. 9, 1096 Budapest, Hungary. 13. MTA-SZTE Research Group for Cardiovascular Pharmacology, Hungarian Academy of Sciences, Dóm tér 12, 6720 Szeged, Hungary.
Abstract
AIMS: Subunit interactions at the cytoplasmic domain interface (CD-I) have recently been shown to control gating in inward rectifier potassium channels. Here we report the novel KCNJ2 variant p.Glu293Lys that has been found in a patient with Andersen-Tawil syndrome type 1 (ATS1), causing amino acid substitution at the CD-I of the inward rectifier potassium channel subunit Kir2.1. Neither has the role of Glu293 in gating control been investigated nor has a pathogenic variant been described at this position. This study aimed to assess the involvement of Glu293 in CD-I subunit interactions and to establish the pathogenic role of the p.Glu293Lys variant in ATS1. METHODS AND RESULTS: The p.Glu293Lys variant produced no current in homomeric form and showed dominant-negative effect over wild-type (WT) subunits. Immunocytochemical labelling showed the p.Glu293Lys subunits to distribute in the subsarcolemmal space. Salt bridge prediction indicated the presence of an intersubunit salt bridge network at the CD-I of Kir2.1, with the involvement of Glu293. Subunit interactions were studied by the NanoLuc® Binary Technology (NanoBiT) split reporter assay. Reporter constructs carrying NanoBiT tags on the intracellular termini produced no bioluminescent signal above background with the p.Glu293Lys variant in homomeric configuration and significantly reduced signals in cells co-expressing WT and p.Glu293Lys subunits simultaneously. Extracellularly presented reporter tags, however, generated comparable bioluminescent signals with heteromeric WT and p.Glu293Lys subunits and with homomeric WT channels. CONCLUSIONS: Loss of function and dominant-negative effect confirm the causative role of p.Glu293Lys in ATS1. Co-assembly of Kir2.1 subunits is impaired in homomeric channels consisting of p.Glu293Lys subunits and is partially rescued in heteromeric complexes of WT and p.Glu293Lys Kir2.1 variants. These data point to an important role of Glu293 in mediating subunit assembly, as well as in gating of Kir2.1 channels. Published on behalf of the European Society of Cardiology. All rights reserved.
AIMS: Subunit interactions at the cytoplasmic domain interface (CD-I) have recently been shown to control gating in inward rectifier potassium channels. Here we report the novel KCNJ2 variant p.Glu293Lys that has been found in a patient with Andersen-Tawil syndrome type 1 (ATS1), causing amino acid substitution at the CD-I of the inward rectifier potassium channel subunit Kir2.1. Neither has the role of Glu293 in gating control been investigated nor has a pathogenic variant been described at this position. This study aimed to assess the involvement of Glu293 in CD-I subunit interactions and to establish the pathogenic role of the p.Glu293Lys variant in ATS1. METHODS AND RESULTS: The p.Glu293Lys variant produced no current in homomeric form and showed dominant-negative effect over wild-type (WT) subunits. Immunocytochemical labelling showed the p.Glu293Lys subunits to distribute in the subsarcolemmal space. Salt bridge prediction indicated the presence of an intersubunit salt bridge network at the CD-I of Kir2.1, with the involvement of Glu293. Subunit interactions were studied by the NanoLuc® Binary Technology (NanoBiT) split reporter assay. Reporter constructs carrying NanoBiT tags on the intracellular termini produced no bioluminescent signal above background with the p.Glu293Lys variant in homomeric configuration and significantly reduced signals in cells co-expressing WT and p.Glu293Lys subunits simultaneously. Extracellularly presented reporter tags, however, generated comparable bioluminescent signals with heteromeric WT and p.Glu293Lys subunits and with homomeric WT channels. CONCLUSIONS: Loss of function and dominant-negative effect confirm the causative role of p.Glu293Lys in ATS1. Co-assembly of Kir2.1 subunits is impaired in homomeric channels consisting of p.Glu293Lys subunits and is partially rescued in heteromeric complexes of WT and p.Glu293Lys Kir2.1 variants. These data point to an important role of Glu293 in mediating subunit assembly, as well as in gating of Kir2.1 channels. Published on behalf of the European Society of Cardiology. All rights reserved.